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BMC Cancer BioMed Central

Research article Open Access


EGFR and HER2 expression in primary cervical cancers and
corresponding lymph node metastases: Implications for targeted
radiotherapy
Li Shen1, Yongjie Shui2, Xiaojia Wang3, Liming Sheng2, Zhengyan Yang3,
Danfeng Xue3 and Qichun Wei*1,2

Address: 1Department of Radiation Oncology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, PR
China, 2Ministry of Education Key Laboratory of Cancer Prevention and Intervention, Zhejiang University School of Medicine, Hangzhou 310009,
PR China and 3Zhejiang Cancer Hospital, Banshan, Hangzhou, PR China
Email: Li Shen - lishenhz@yahoo.com; Yongjie Shui - yjshuihz@yahoo.com; Xiaojia Wang - xjwangzj@tom.com;
Liming Sheng - lmshenghz@yahoo.com; Zhengyan Yang - zyyangzj@yahoo.com; Danfeng Xue - dfxuezj@yahoo.com;
Qichun Wei* - Qichun_Wei@zju.edu.cn
* Corresponding author

Published: 12 August 2008 Received: 22 March 2008


Accepted: 12 August 2008
BMC Cancer 2008, 8:232 doi:10.1186/1471-2407-8-232
This article is available from: http://www.biomedcentral.com/1471-2407/8/232
© 2008 Shen et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Proteins overexpressed on the surface of tumor cells can be selectively targeted.
Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2)
are among the most often targeted proteins. The level and stability of expression in both primary
tumors and corresponding metastases is crucial in the assessment of a receptor as target for
imaging in nuclear medicine and for various forms of therapy. So far, the expression of EGFR and
HER2 has only been determined in primary cervical cancers, and we have not found published data
regarding the receptor status in corresponding metastatic lesions. The goal of this study was to
evaluate whether any of these receptors are suitable as target for clinical diagnosis and therapy.
Methods: Expression of EGFR and HER2 was investigated immunohistochemically in both lymph
node metastases and corresponding primary cervical cancers (n = 53). HER2 and EGFR expression
was scored using HercepTest criteria (0, 1+, 2+ or 3+).
Results: EGFR overexpression (2+ or 3+) was found in 64% (35/53) of the primary cervical tumors
and 60% (32/53) of the corresponding lymph node metastases. There was a good concordance
between the primary tumors and the paired metastases regarding EGFR expression. Only four
patients who had 2+ or 3+ in the primary tumors changed to 0 or 1+ in lymph node metastases,
and another two cases changed the other way around. None of the primary tumors or the lymph
node metastases expressed HER2 protein.
Conclusion: The EGFR expression seems to be common and stable during cervical cancer
metastasis, which is encouraging for testing of EGFR targeted radiotherapy. HER2 appears to be of
poor interest as a potential target in the treatment of cervical cancer.

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Background static cervical cancer were treated with gefitinib 500 mg/
Cervical cancer represents the second most frequent day, EGFR expression levels by means of immunohisto-
malignancy in women worldwide, particularly in devel- chemistry did not correlate with tumor response and dis-
oping countries [1]. While curable in early stages, the ease control [20]. This is not unexpected, since EGFR
prognosis for advanced stage disease is poor [2]. Radia- expression does not necessarily correlate with EGFR recep-
tion has been the gold standard of therapy for many dec- tor activation. Other molecular alterations, such as EGFR
ades. Nowadays, concurrent cisplatin-based gene amplification, mutations of the tyrosine kinase
chemoradiotherapy has been considered as the standard domain, and EGFR phosphorylation status, might be use-
therapeutic modality for locally advanced cervical cancer ful indicators for the response to EGFR signaling inhibi-
[3-6]. However, such treatment remains suboptimal with tion. However, in the case of targeted radionuclide
histopathological residual tumor observed in 40–50% of therapy, tumor cells are mainly killed with delivered radi-
patients [7,8]. Those presenting with recurrent or meta- ation and therapeutic efficiency is only dependent on the
static disease have limited treatment options [2,9], and receptor expression and not whether the receptor function
the 5-year survival is less than 5% [10]. There is a clear can be blocked or not. Thus, receptor overexpression is
need for novel, more effective therapeutical strategies to considered necessary for the success of targeted radiother-
improve overall survival and the quality of life for apy.
advanced, recurrent and disseminated cervical cancer.
Thus, the testing of molecular targeted therapies against Previous studies have shown EGFR to be frequently
cervical cancer is a logical step to follow [11]. Another expressed in primary cervical cancer [21-25], and that
strategy is receptor-mediated tumor targeted radiotherapy EGFR expression is correlated with poor prognosis [25-
[12], which is based on the delivery of therapeutically rel- 27]. Positive staining of HER2 in cervical cancer has been
evant radionuclides directly to disseminated tumor cells, reported to vary wildly in the earlier studies [28-30]. How-
with hopefully minimal damage to normal tissues. ever, according to the recent reports using HercepTest
grading system, HER2 overexpression was considered to
Proteins overexpressed on the surface of tumor cells can be a rare event in primary cervical cancer [31,32].
be selectively targeted. Epidermal growth factor receptor
(EGFR) and human epidermal growth factor receptor 2 The literature was reviewed for similar investigations, but
(HER2) are among the most often targeted proteins. The no other studies comparing primary cervical cancers and
presence of EGFR and HER2 receptors have been associ- their corresponding metastases regarding EGFR and HER2
ated with accelerated tumor progression and therapeutic were found by the authors. It is still unclear whether the
resistance for several types of malignancies, including cer- metastases lose, gain, or retain the receptor status relative
vical cancer. The causal relationship of EGFR and HER2 to the primary tumors. For a receptor to be of interest for
receptor network to disease progression and resistance to targeting, similar expression in both the primary tumors
therapy provides a rationale for therapeutic intervention. and the disseminated lesions are required. Investigation
into the receptor status between metastases and the pri-
Nowadays, EGFR targeted drugs, both chimeric mono- mary tumors will provide valuable information on
clonal antibody Cetuximab (Erbitux) and the small-mol- whether these receptors are suitable as target for diagnos-
ecule tyrosine kinase inhibitors (e.g. Iressa and Tarceva) tic and/or therapeutic procedures. In the present study,
[13-15], have been approved by FDA. Clinical trials of the expression of EGFR and HER2 was investigated immu-
Cetuximab, either alone or in combination with radio- nohistochemically in a series of 53 primary cervical can-
therapy/chemotherapy, have recently demonstrated effi- cers and corresponding lymph node metastases.
cacy in patients with head and neck cancer, colorectal
cancer and lung cancer [16-18]. The humanized mono- Methods
clonal antibody trastuzumab (Herceptin), which is the Patients and Samples
first clinically available oncogene-targeted therapeutic Patients with cervical cancer who were treated with radical
agent for treatment of solid tumors, has boosted the inter- hysterectomy and systematic pelvic lymphadenectomy
est of physicians in targeting therapy, as therapeutic bene- between 2002 and 2003, were enrolled in the present
fit was proved in patients with HER2-positive metastatic study. Tumor samples from all patients were obtained at
breast cancer [19]. On the basis of the preliminary success, the time of operation through the Gynecologic Oncology
clinical trials are currently investigating the therapeutic Department and the Pathology Department, Zhejiang
potential of molecular targeted drugs in other human Cancer Hospital, under ethical approval of the Institu-
malignancies including cervical cancer [20]. tional Review Board of Zhejiang Cancer Hospital.
Informed consent for scientific evaluation had been
In a recent phase II trial reported by Goncalves et al, obtained from patients. Paraffin sections from both the
patients with recurring locoregionally advanced or meta- primary tumors and the corresponding lymph node

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metastases were required for inclusion. Tissue samples endogenous peroxidase was blocked in 0.3% H2O2 in PBS
were not taken from distant metastases so these were not for 20 min. For antigen retrieval, the sections were submit-
available for analysis. Totally, 53 patients with high qual- ted to high temperature and pressure with Tris-EDTA
ity material were finally included in the study. Clinical buffer (pH 9) for 5 min. The slides were preincubated in
information was obtained from the hospital records and PBS for 10 min. The primary mouse monoclonal antibody
included patient age, disease stage, histological pattern, directed against EGF receptor (clone 31G7, Zymed labs,
differentiation, tumor size, nodal involvement, lym- South San Francisco, CA, USA) receptor were diluted
phatic/vascular invasion, vaginal invasion, parametrial 1:100, and incubated overnight at 4°C. The secondary
invasion, ovarian metastasis. All patients had a pathologic biotinylated antibodies (goat anti-mouse from Dako,
diagnosis of squamous cell carcinoma. Ovarian metastasis Glostrup, Denmark) and the peroxidase-labelled strepta-
was not found in this set of cases. The patient and tumor vidin-biotin complex (Dako) were diluted 1:200 and
characteristics of the analyzed cases are shown in Table 1. incubated for 30 min at room temperature. All slides were
developed in 0.05% diamino benzidine (Sigma, St Louis,
Briefly, the tissues were fixed in 4% buffered formalin, MO, USA) for 5 min and counterstained in Harris haema-
processed and embedded in paraffin. Sections, 4-μm toxylin (Sigma). Finally, the slides were dehydrated
thick, were then cut and deparaffinized in xylene and through graded alcohol to xylene and mounted in organic
hydrated through graded concentrations of ethanol to dis- mounting medium.
tilled water.
HER2-staining
EGFR-staining The HER2 immunohistochemical staining was made as
EGFR was assessed by immunohistochemistry using a previously described [34]. After deparaffinization, the sec-
streptavidin-biotin complex technique as previously tions were incubated in methanol and hydrogen peroxide
described [33]. After deparaffinization of the sections, for 30 min quenching endogenous peroxidase. Antigen
retrieval was done in waterbath at 98°C, pH 6 for 40 min-
Table 1: Tumor and patient characteristics (n = 53) utes. Thereafter the slides were cooled at room tempera-
Characteristics Patients, n (%) ture and then washed in distilled water.
Immunohistochemical staining was performed using the
Differentiation Elite ABC Kit (Vectastain, Vector Laboratories, Burlin-
High 5 (9.4) game, CA). Blocking serum was applied for 15 min and
Moderate 42 (79.3) followed by incubation with rabbit anti-human c-erbB-2
Low 6 (11.3) oncoprotein (code No. A 0485, Dako) diluted 1:350. Sec-
tions were then incubated with the biotinylated secondary
FIGO Stage
IB 16 (30.2)
antibody and were visualised by using the peroxidase sub-
IIA 35 (66.0) strate 3-amino-9-ethyl-carbazole (AEC) (Sigma A-5754)
IIB 1 (1.9) as chromogen. Finally, the sections were counterstained
IIIB 1 (1.9) with Mayer's haematoxylin and mounted.

Tumor size EGFR and HER2-scores


> 5 cm 9 (17.0)
The HER2 expression was scored using the HercepTest
3–5 cm 26 (49.0)
< 3 cm 18 (34.0) scoring criterion. The HER2-score was based on a scale
where 0 corresponded to tumor cells that were completely
Lymphatic or vascular space invasion negative, 1+ corresponded to faint perceptible staining of
Positive 26 (49.1) the tumor cell membranes, 2+ corresponded to moderate
Negative 27 (50.9) staining of the entire tumor cell membranes and 3+ was
strong circumferential staining of the entire tumor cell
Vaginal invasion
membranes creating a fishnet pattern. The Canadian and
Positive 37 (69.8)
Negative 16 (30.2) the DAKO HercepTest guidelines were applied, which
require more than 10% of the tumor cells to be stained
Parametrial invasion [35]. Cytoplasmic staining was considered non-specific
Positive 6 (11.3) and was not included in the scoring. As positive controls
Negative 47 (88.7) we used in house positive control tissue sections as well as
positive control sections supplied by DAKO. As negative
Age (years)
controls we used normal tissues, which are expected not
Medium 48
Range 29–72 to express HER2 such as connective tissue seen in the same
sections as the tumor cells. In the metastases sections we

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used lymphocytes and the surrounding capsule of the sponding lymph node metastases in the majority of cases.
lymph nodes as negative internal controls. The expression Fifteen changes were observed. However, there were only
pattern of EGFR is quite similar to that of HER2, and EGFR four patients who had 2+ or 3+ in the primary tumors and
expression was therefore evaluated using the same scoring changed to 0 or 1+ in lymph node metastases, and
criterion as for HER2. As EGFR positive controls we used another two patients who had 0 or 1+ in the primary
in house positive control skin tissue sections. As negative tumors and changed to 2+ or 3+ in lymph node metas-
controls we used connective tissue seen in the same sec- tases. Examples of staining patterns for a primary tumor
tions as the tumor cells. In the metastases sections we used and the corresponding metastasis (which both were
lymphocytes and the surrounding capsule of the lymph scored as 3+) are shown in Figure 1A and 1B.
nodes as negative internal controls.
Expression of HER2
Results Among the 53 paired samples, none of the primary
Expression of EGFR tumors or the lymph node metastases expressed HER2
Table 2 shows the EGFR-scores for the analyzed 53 pri- protein. In fact, a few strong membrane stained tumor
mary cervical squamous cell carcinoma and the corre- cells were seen in 2 cases, but the stained cells were less
sponding 53 lymph node metastases. EGFR than 10% of total tumor cells (Figure 2). So, these two
overexpression (2+ or 3+) was found in 64% (34/53) of cases were also interpreted as negative.
the primary cervical tumors and 60% (32/53) of the cor-
responding lymph node metastases. There was a good
agreement between the primary tumors and the corre-

Figure 1
Comparisons of immunohistochemical EGFR staining of primary cervical carcinoma (A) and corresponding metastases (B)
Comparisons of immunohistochemical EGFR staining of primary cervical carcinoma (A) and corresponding
metastases (B). Both A and B (from the same patient) were scored 3+. The micrographs were taken with objective × 40.

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Table 2: EGFR-scores for the analyzed primary cervical was to evaluate whether any of these receptors are suitable
squamous cell carcinoma and the corresponding lymph node as target for radionuclide based imaging and radiation
metastases (n = 53)
therapy.
Primary tumor EGFR-scores Lymph node metastases EGFR-scores
0 1+ 2+ 3+ Overexpression of EGFR in cervical cancer has been
reported to be common (ranges from 26–72%) [21-25].
0 5 1 0 0 However, it is unclear whether metastases lose, gain, or
1+ 1 10 2 0 retain EGFR status relative to the primary cervical tumors.
2+ 0 3 13 3
Studies on the EGFR status of metastatic lymph node of
3+ 1 0 4 10
cervical cancer will provide precious knowledge to evalu-
The scoring was based on a scale where 0 corresponded to ate whether the receptor is of interest for diagnostic and/
completely negative staining, 1+ corresponded to faint perceptible or therapeutic procedures or not.
staining of the tumor cell membranes, 2+ corresponded to moderate
staining of the entire tumor cell membranes and 3+ was strong EGFR overexpression (2+/3+) was found in 64% of the
circumferential staining of the entire tumor cell membranes creating a
fishnet pattern primary lesions. Our result is consistent with the former
findings of high EGFR overexpression in cervical cancer
Discussion [23-25]. Furthermore, we found that the frequency of
The aim of this study was to evaluate the EGFR and HER2 EGFR overexpression in lymph node metastases was
receptor expression, using immunohistochemical analy- approximately as high as in the primary lesions of cervical
ses, in primary cervical cancers and determine if the cancer. Although 15 changes were observed, only 4
expression is retained in lymph node metastases. The goal patients with EGFR overexpression in the primary tumor
had lower EGFR scores in the corresponding lymph node
metastases. Moreover, in another two patients, EGFR
overexpression was gained in lymph node metastases
while the primary tumors had low scores. Actually, immu-
nohistochemistry is not a strickly quantitative method.
Methodological differences, e.g. in fixation procedures,
retrieval methods, and antibodies, are likely to affect the
sensitivity of EGFR staining. In addition, the criteria for
defining positive EGFR expression could also account for
part of the discrepancies between the primaries and
metastases. For example, if membrane staining in more
than 10% of the tumor cells was defined as positive EGFR
expression, regardless of pattern of cellular membrane
staining (complete or incomplete), only 3 changes could
be observed: two cases from positive to negative when the
primary lesions were compared to the corresponding
lymph node metastases, and one case changed the other
way around. Nevertheless, it seems that, in a majority of
the studied cases, EGFR expression retained in the metas-
tases.

To our knowledge, the question of EGFR status in lymph


node metastases versus primary cervical cancer has not
been addressed. From our results, it seems that EGFR
expression is stable when comparing the lymph node
metastases with the primary cervical cancers, which is sur-
prising in the light of the genomic instability that charac-
terizes most malignant tumors. Although the current
Figure
Example
cervical
10% of total
2carcinoma,
of immunohistochemical
tumor scored
cells) 0 (stained
HER2
cells
staining
were less
of primary
than report is limited by the small sample size, our observa-
Example of immunohistochemical HER2 staining of tions suggest that EGFR expression in the primary tumors,
primary cervical carcinoma, scored 0 (stained cells which can readily be determined after surgery or biopsy,
were less than 10% of total tumor cells). This case also might predict EGFR-positive metastases with a reasonably
had negative HER2 staining in lymph node metastasis. The
high probability.
micrograph was taken with objective × 10.

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In EGFR targeted radionuclide therapy, possible side Authors' contributions


effects to normal tissues should be taken into considera- LS participated in the design of the study, carried out the
tion, as EGFR is commonly expressed in normal cells. It clinical and immunohistochemical data analysis; YS and
might be possible to minimize the toxicity and improve LS interpreted the histological and immunohistochemical
therapeutic efficiency by using suitable targeting agents data; XW, ZY and DX contribute with the clinical data; and
with low uptake in critical normal tissues, and suitable QW conceived the study, interpreted the immunohisto-
biodistribution. EGFR targeted radiotherapy might also chemical data and wrote the manuscript. All authors read
be possible if a tumor and its metastases have a strong and approved the final manuscript.
EGFR expression to ensure higher tumor uptake than in
most normal tissues or local delivery of the targeting agent Acknowledgements
can be made. The authors thank Min Lin for help with the immunohistochemical stainings
and Xingguo Lu for help with the photos in Figure 1 and 2. The authors
There are conflicting results regarding the frequency of acknowledge economical support from a grant from National Natural Sci-
ence Foundation of China to Q Wei (No. 30470501).
HER2 expression in cervical cancer. High positive rate of
HER2 expression in cervical cancer has been reported in
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T, Paterson M, Sharp F, Cruse P: The significance of Erb-b2 Your research papers will be:
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